Coxiella burnetii is the causative agent of human Q fever, an emerging infectious disease and a leading cause of culture-negative endocarditis. An obligate intracellular bacterial pathogen spread through aerosols, Coxiella primarily targets alveolar macrophages during natural infection. Once inside the cell, Coxiella manipulates host cell machinery to promote the biogenesis of a specialized compartment called the parasitophorous vacuole (PV). While the PV is central to Coxiella pathogenesis, the mechanisms behind PV biogenesis and maintenance are poorly understood. The Coxiella PV is sterol-rich, and pharmaceutical inhibitors that perturb host cell cholesterol inhibit PV formation and subsequent bacterial growth. Despite increasing evidence that cholesterol plays a critical role in PV formation and Coxiella-host interactions, there is very little information on either the bacterial or host factors involved. The objective of this application is to identify and characterie bacterial-driven changes in host cell cholesterol homeostasis. This proposal will test the hypothesis that Coxiella proteins, secreted into the host cell cytoplasm through the bacteria's specialized Type IV Secretion System (T4SS), manipulate host cholesterol homeostasis as a mechanism to support intracellular bacterial growth and survival. In the first specific aim, host cholesterol homeostasis pathways targeted by Coxiella T4SS effector proteins will be identified through transcriptome analysis of infected alveolar macrophages. This study will identify key host cell genes differentially regulated by Coxiella. Following their identification, the host pathways will be disrupted and the effect on Coxiella survival determined. In the second aim, Coxiella proteins that manipulate host cholesterol will be characterized. This will be accomplished by screening for Coxiella transposon mutants that have lost the ability to alter host cell cholesterol homeostasis. The identified Coxiella proteins will be characterized by analyzing the phenotype of the isolated mutants, as well as functional analysis of the corresponding protein. The proposed work will identify both host and pathogen proteins involved in cholesterol homeostasis during Coxiella infection, opening new avenues for therapeutic intervention.